LiOAcYEASTTRANSFORMATION^a

(seenotesatend)

UseSTERILETECHNIQUEthroughout.

1.Inoculatea10mlovernightculturewithasingleyeastcolony.Ifstartingwithayeaststrainwithnoplasmids,useYPD.Ifstartingwithastrainthatalreadyhasoneormoreplasmidsuseliquiddrop-outmedialackingtheappropriatenutrient.^bIncubateat30^oCinrollerdrumattopspeedovernight.^b

2.MeasuretheOD₆₀₀oftheovernightculture.^cMakeanappropriatedilutionoftheovernightcultureinto50mlofmediainasterile250mlflasksothatthefinalOD₆₀₀is0.2.

3.Incubateat30^oC,shakingat200rpm,untilOD₆₀₀=1.0

4.Transferculture(usingsteriletechnique!)toasterile50mlpolypropylenetube(Oakridgetube).Spininclinicalcentrifugeat3000rpm,2min.Thisandallfollowingmanipulationsaredoneatroomtemperature.

5.Dumpoffmedia(quicklyandcarefully)andadd10mlsterilewater.ResUSPendcellsbyswirlingorgentlevortexing.

6.Pelletcellsinclinicalcentrifugeat3000rpm,2min.Pouroffwater.

7.Add5mlofLiOAc/TEsolution.Resuspendcellsbyswirlingorgentlevortexing.

8.Pelletcellsinclinicalcentrifugeat3000rpm,2min.PouroffLiOAc/TE.

9.Add0.25mlofLiOAc/TEsolution.Resuspendcellsbyswirlingorgentlevortexing.

10.Foreachtransformationaliquot50µlofcellstoasterileEppendorftube.ALWAYSsetupacontroltubethatwillgetnoDNA.

11.AddplasmidDNA^d.[Forhigherefficiency(e.g.forlibrarytransformations^e)addDMSOto10%(e.g.~6µl)].Mixgently.Add300µl40%PEGinLiOAc/TE.Mixbyinvertingtwoorthreetimes.

12.Donotvortex.InthepresenceofPEGyeastcellsaremorefragile.

13.Incubateat30^oCfor30min.Takethistimetocheckthetemperatureofthe42^oC,andwarmandlabelplates.Removecellstoroomtemperature.

14.Incubateat42^oCforexactly15min.Removetoroomtemperature.

15.Plate200-300µl,includingthebulkofthecells,ontoappropriateplates.Incubateat30^oCwithplatesrightsideup.Turnplatesoverafter1day.

NOTES

^aThismethodisbasedontheprocedureofGeitzetal.,1992NucleicAcidsResearch20:1425.

SeealsoGeitzLabYeastTransformationHomePage

^bWhenusingYPD,12to16hoursissufficientfora10mlovernightculture.Growthindrop-outmediamaytakemuchlongersothe"overnight"culturemaybestartedinthemorningforthefollowingday.Alternatively,a50mlovernightculturemaybestarted-whichmayormaynotneedtobedilutedthenextday.

^cSpectrophotometermeasurementsareonlylinearatOD₆₀₀between~0.05and1.0.IftheOD₆₀₀ofthecultureisover1.0itshouldbediluted10-foldtotakethemeasurement.Note:IdeallyoneshouldtrytransformingyeastatdifferentOD₆₀₀from0.7to1.5toseewhichisbest.Spectscandiffer,andthisapproachiseasierthancountingcells

^dUsenomorethan1µgplasmidDNA/50µlofcells.Forroutinetransformations,5-10µlofminiprepDNAor0.1-0.5µgmaxiprepDNAissufficient.Forlibrarytransformationsuse1µgplasmidPLUS30µgcarrierDNA(suchasBoehringerMBgradefishspermDNACat#1467140;seealsoSchiestlandGietz(1989)CurrentGenetics16:339-346).

^eToperform10ormoretransfromations,scaleupbyusinga200mlcultureatstep2,andresuspendin1mlLiOAc/TEsolutionatstep9.

SOLUTIONS

LiOAc/TE

For50ml,inasterile50mlFalcontube.

44.4mlsterilewater

5.0mlsterile1MLiOAc,pH7.0-7.4

0.5mlsterile1MTrisHCl,pH7.5

0.1mlsterile500mMEDTA

40%PEGinLiOAc/TE

For50ml,inasterile50mlFalcontube.

40mlsterile50%PEG4000

5.0mlsterile1MLiOAc,pH7.0-7.4

0.5mlsterile1MTrisHCl,pH7.5

0.1mlsterile500mMEDTA

4.4mlsterilewater.

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